Integrin Regulation of IGF-1 Responsiveness in Bone During Mechanical Loading

描述(由申请人提供)：大量证据支持IGF-I信号在调节骨骼对负荷的反应中的作用。骨细胞和成熟的成骨细胞可能是机械负荷最初传递为新骨形成信号的候选细胞。在机械负荷后，这些细胞中IGF-I的产生增加，这种IGF-I可能是产生新的成骨细胞的信号。此外，机械负荷增强了骨骼对IGF-I的反应，就像骨骼卸载导致对IGF-I在骨形成和骨祖细胞增殖方面的抵抗一样。这种在体内或体外卸载时对IGF-I的反应失败是由于IGF-I未能激活IGF-I受体(IGF-IR)，尽管IGF-IR水平和IGF-I与其受体的结合是正常的。通过在体内或体外对成骨细胞重新加载，可以逆转骨骼卸载引起的对IGF-I的抵抗。整合素在这一过程中发挥着重要作用。骨骼卸载减少了整合素的表达，这种表达的抑制可以通过重新加载骨骼来恢复，这与IGF-I反应的恢复是相称的。免疫共沉淀和共聚焦研究表明整合素1和3亚基直接与IGF-IR结合，IGF-I能增强这种结合。生长在特定整合素底物上的细胞表现出IGF-I和负载对IGF-IR的激活增强，以及IGF-I对整合素亚单位的激活增强。成骨细胞中整合素1或3亚单位的敲除阻断了IGF-I激活其受体的能力，并通过加载伴随着IGF-I下游信号中断而阻止其激活。因此，IGF-I信号对于骨骼对骨的反应是重要的，该信号是由整合素调节的。在这个项目中，我们将检验这样一个假设，即骨对机械负荷的合成代谢反应需要整合素和IGF-I信号之间的协同作用。提出了三个目标：1.确定整合素在机械负荷过程中对IGF-I信号的调节机制，以及成骨细胞的成熟对这一调节的调节程度。这些研究将在脉动流体流动(PFF)机械加载的成骨细胞和骨细胞系中进行，分析整合素/IGF-IR复合体的组成和作用。2.确定骨骼对机械负荷反应的IGF-I和IGF-IR的需要量。这些研究将在从成骨细胞和成骨细胞/骨细胞中删除IGF-I和IGF-IR的小鼠身上进行，然后在体内和体外测定对负荷的反应。3.确定骨骼对机械负荷反应中整合素1和3亚基的需要量。这些研究将通过从成骨细胞和成骨细胞/骨细胞中删除1和3整合素，然后确定它们对负荷和IGF-I的反应来完成。因此，使用体内和体外方法，我们将确定整合素/IGF-I信号在骨骼对负荷的反应中的作用和重要性，结果对于我们理解骨骼固定过程中的骨丢失至关重要，这些原因包括瘫痪和受伤或疾病后的长时间制动。公共卫生相关性：骨骼卸载导致骨骼丢失，部分原因是骨骼不能对IGF-1的合成代谢作用作出反应。这种失败与整合素表达的丧失有关。本项目将评估整合素和IGF-1信号在骨骼对机械负荷的反应中的相互作用。

DESCRIPTION (provided by applicant): Substantial evidence supports the role for IGF-I signaling in mediating the skeletal response to load. Osteocytes and mature osteoblasts are the likely candidates by which mechanical load is initially transmitted into signals for new bone formation. IGF-I production is increased in these cells following mechanical load, and this IGF-I may be the signal for the production of new osteoblasts. Furthermore, mechanical load enhances the skeletal response to IGF-I just as skeletal unloading leads to resistance to IGF-I with respect to bone formation and osteoprogenitor proliferation. This failure to respond to IGF-I in vivo or in vitro with unloading is due to a failure of IGF-I to activate the IGF-I receptor (IGF-IR), although IGF-IR levels and binding of IGF-I to its receptor are normal. The resistance to IGF-I caused by skeletal unloading can be reversed by reloading the bones in vivo or the osteoblasts in vitro. Integrins play an important role in this process. Skeletal unloading reduces integrin expression, and this inhibition of expression can be restored by reloading the bones commensurate with restoration of IGF-I responsiveness. Co-immunoprecipitation and confocal studies demonstrate direct binding of ¿1 and ¿3 integrin subunits to the IGF-IR, binding which is enhanced by IGF-I. Cells grown on specific integrin substrates demonstrate enhanced activation of the IGF-IR by both IGF-I and loading, and enhanced IGF-I activation of the ¿3 integrin subunit. Knockdown of either the ¿1 or ¿3 integrin subunits in osteoblasts blocks the ability of IGF-I to activate its receptor and prevents its activation by loading concomitant with a disruption of downstream IGF-I signaling. Thus, IGF-I signaling is important for the skeletal response to bone, and this signaling is regulated by integrins. In this project we will test the hypothesis that the anabolic response of bone to mechanical load requires the synergistic interaction between integrin and IGF-I signaling. Three aims are proposed: 1. Determine the mechanism for regulation of IGF-I signaling by integrins during mechanical loading, and the extent to which the maturation of the osteoblast modifies this regulation. These studies will be done with osteoblast and osteocyte cell lines mechanically loaded by pulsatile fluid flow (PFF), analyzing the components and roles of the integrin/IGF-IR complex. 2. Determine the requirement for IGF-I and IGF-IR in the skeletal response to mechanical load. These studies will be done with mice in which IGF-I and IGF-IR are deleted from osteoprogenitors and osteoblasts/osteocytes then determining the response to load in vivo and in vitro. 3. Determine the requirement for the ¿1 and ¿3 integrin subunits in the skeletal response to mechanical load. These studies will be done by deleting ¿1 and ¿3 integrin from osteoprogenitors and osteoblasts/osteocytes then determining their response to load and to IGF-I. Thus using both in vivo and in vitro approaches we will establish the role and importance of integrin/IGF-I signaling in the skeletal response to load, results critical to our understanding of bone loss during skeletal immobilization from a variety of causes including paralysis and prolonged immobilization following injury or sickness. PUBLIC HEALTH RELEVANCE: Skeletal unloading results in the loss of bone which is due in part to failure of bone to respond to the anabolic actions of IGF-1. This failure is associated with loss of integrin expression. This project will evaluate the interaction between integrin and IGF-1 signaling in the skeletal response to mechanical load.